Human amniotic fluid stem cell injection therapy for urethral sphincter regeneration in an animal model

ABSTRACT: BACKGROUND: Stem cell injection therapies have been proposed to overcome the limited efficacy and adverse reactions of bulking agents. However, most have significant limitations, including painful procurement, requirement for anesthesia, donor site infection and a frequently low cell yield. Recently, human amniotic fluid stem cells (hAFSCs) have been proposed as an ideal cell therapy source. In this study, we investigated whether periurethral injection of hAFSCs can restore urethral sphincter competency in a mouse model. METHODS: Amniotic fluids were collected and harvested cells were analyzed for stem cell characteristics and in vitro myogenic differentiation potency. Mice underwent bilateral pudendal nerve transection to generate a stress urinary incontinence (SUI) model and received either periurethral injection of hAFSCs, periurethral injection of Plasma-Lyte (control group), or underwent a sham (normal control group). For in vivo cell tracking, cells were labeled with silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate (MNPs@SiO2 (RITC)) and were injected into the urethral sphincter region (n = 9). Signals were detected by optical imaging. Leak point pressure and closing pressure were recorded serially after injection. Tumorigenicity of hAFSCs was evaluated by implanting hAFSCs into the subcapsular space of the kidney, followed two weeks later by retrieval and histologic analysis. RESULTS: Flow activated cell sorting showed that hAFSCs expressed mesenchymal stem cell (MSC) markers, but no hematopoietic stem cell markers. Induction of myogenic differentiation in the hAFSCs resulted in expression of PAX7 and MYOD at Day 3, and DYSTROPHIN at Day 7. The nanoparticle-labeled hAFSCs could be tracked in vivo with optical imaging for up to 10 days after injection. Four weeks after injection, the mean LPP and CP were significantly increased in the hAFSC-injected group compared with the control group. Nerve regeneration and neuromuscular junction formation of injected hAFSCs in vivo was confirmed with expression of neuronal markers and acetylcholine receptor. Injection of hAFSCs caused no in vivo host CD8 lymphocyte aggregation or tumor formation. CONCLUSIONS: hAFSCs displayed MSC characteristics and could differentiate into cells of myogenic lineage. Periurethral injection of hAFSCs into an SUI animal model restored the urethral sphincter to apparently normal histology and function, in absence of immunogenicity and tumorigenicity.     

Human amniotic fluid CX-L-fucosidase

The activity and properties of alpha-L-fucosidase in 24 samples of amniotic fluid have been investigated using the 4-methylumbelliferyl substrate. A wide range of fucosidase specific activity (0.20-1.45 nmol/min/mg protein) was found with an average value of 0.62 nmol/min/mg protein. Although no clear-cut correlation exist, there appear to be trends of decreasing fucosidase specific activity with increasing maternal age of donor and increasing gestational time. alpha-L-Fucosidase activity from four amniotic fluids was characterized kinetically and immunochemically and found not to differ in its properties due to maternal age of donor or gestational time. Isoelectric focusing revealed multiple forms between pH 5.0 and 6.8, with the majority of activity present between pH 5.0 and 6.0. The pH optimum is near pH 5.0 with a second optimum suggested at pH 6.2. The apparent Michaelis constant (Km) for the 4-methylumbelliferyl substrate is 0.05 +/- 0.01 mM. The enzyme is completely thermostable at 37 degrees C for at least 4 h but loses 95% of its activity after preincubation at 45 degrees C for 1 h. Double immunodiffusion and immunoprecipitation experiments using anti-liver alpha-L-fucosidase antibody suggest that the amniotic fluid alpha-L-fucosidase is antigenically similar, if not identical, to the human liver enzyme.     

Human amniotic fluid stem cells support undifferentiated propagation and pluripotency of human embryonic stem cell without b-FGF in a density dependent manner

Human embryonic stem cells (hESCs) are pluripotent cells which can give rise to almost all adult cell lineages. Culture system of hESCs is complex, requiring exogenous b-FGF and feeder cell layer. Human mesenchymal stem cells (MSCs) not only synthesize soluble cytokines or factors such as b-FGF, but also provide other mechanism which might play positive role on sustaining hESCs propagation and pluripotency. Human amniotic fluid stem (AFS) cells, which share characteristics of both embryonic and adult stem cells, have been regarded as promising cells for regenerative medicine. Taking advantage by AFS cells, we studied the ability of AFS cells in supporting undifferentiated propagation and pluripotency of Chinese population derived X-01 hESCs. Human AF-type amniotic fluid stem cells (hAF-AFSCs) transcribed genes including Activin A, TGF-β1, Noggin and b-FGF, which involved in maintaining pluripotency and self-renewal of hESCs. Compared to mouse embryonic fibroblasts (MEFs), hAF-AFSCs secreted higher concentration of b-FGF which was important in hESCs culture (P < 0.05). The hESCs were propagated more than 30 passages on hAF-AFSCs layer with exogenous b-FGF supplementation, keeping undifferentiated status. While exogenous b-FGF was obviated, propagation of hESCs with undifferentiated status was dependent on density of hAF-AFSC feeder layer. Lower density of hAF-AFSCs resulted in rapid decline in undifferentiated clone number, while higher ones hindered the growth of colonies. The most appropriate hAF-AFSCs feeder density to maintain the X-01 hESC line without exogenous b-FGF was 15-20×10⁴/well. To the best of our knowledge, this is the first study demonstrating that hAF-AFSCs could support undifferentiated propagation and pluripotency of Chinese population derived hESCs without exogenous b-FGF supplementation.     

人羊膜间充质干细胞移植修复肝脏缺血-再灌注损伤

BACKGROUND:Studies have shown that human amniotic mesenchymal stem cells can differentiate into hepatocyte-like cells, suggesting that human amniotic mesenchymal stem cell transplantation provides a new potential for the clinical treatment of liver diseases. OBJECTIVE:To observe the effect of human amniotic mesenchymal stem cell transplantation on the repair of liver ischemia-reperfusion injury repair. METHODS:Sixty Sprague-Dawley rats were randomized into stem cell transplantation, model and control groups. Animal models of liver ischemia-reperfusion injury were made in the rats in the stem cell transplantation and model groups. One hour after modeling, rats in the stem cell transplantation were given injection of human amniotic mesenchymal stem cells (0.5 mL, 106 cells) via the tail vein, while rats in the model and control group were given L-DMEM (0.5 mL) or normal saline (0.5 mL), respectively. Liver function and liver morphology were detected at 1, 2, 3 weeks after transplantation. Meanwhile, RT-PCR detection and western blot assay were also conducted. RESULTS AND CONCLUSION:(1) Liver function: Compared with the control group, levels of aspartate aminotransferase, alanine aminotransferase and malondialdehyde were significantly increased in the model group at different time points after transplantation (P < 0.05), while a significant reduction in the levels of these three indicators was found after cell transplantation as compared with the model group (P < 0.05). (2) Liver morphology: 2 weeks after transplantation, rats in the model group exhibited hepatocyte degeneration and necrosis, and severe fibrosis, but these changes were remarkably alleviated in the stem cell transplantation group. (3) PT-PCR and western blot detection: 2 weeks after transplantation, a significantly higher level of hepatocyte growth factor in the liver tissue and a lower level of α-smooth muscle protein were found in the stem cell transplantation group compared with the model group (P < 0.05). All these experimental findings indicate that human amniotic mesenchymal stem cell transplantation can improve impaired liver function in rats, possibly through regulating hepatocyte growth factor and α-smooth muscle protein expression levels in the liver, and thereby promotes the repair of liver ischemia-reperfusion injury.     

Chromosomal mosaicism in amniotic fluid cell cultures

Six cases of chromosomal mosaicism detected in amniotic fluid cultures are described. In five of these there was no evidence of fetal mosaicism. In one case fetal mosaicism was demonstrated but only by the study of fibroblasts since blood cultures showed only normal cells. The implications of amniotic fluid mosaicism are discussed and it is concluded that this usually does not indicate fetal mosaicism. The value of repeated amniocentesis in the diagnosis of fetal mosaicism was demonstrated by findings in three of the cases. It is recommended that amniotic fluid cultures be harvested in situ for chromosome studies and that cytogenetic results be expressed as number of colonies karyotyped rather than as number of cells analyzed.     

Anti-HLA antibodies in regenerative medicine stem cell therapy

Research on stem cell therapies for regenerative medicine is progressing rapidly. Although the use of autologous stem cells is a tempting choice, there are several instances in which they are either defective or not available in due time. Allogenic stem cells derived from healthy donors presents a promising alternative. Whether autologous or allogenic, recent advances have proven that stem cells are not as immune privileged as they were thought. Therefore understanding the interactions of these cells with the recipient immune system is paramount to their clinical application. Transplantation of stem cells induces humoral as well as cellular immune response. This review focuses on the humoral response elicited by stem cells upon their administration and consequences on the survival and maintenance of the graft. Current transplantation identifies pre- and post-transplantation anti-HLA antibodies as immune rejection and cell signaling effectors. These two mechanisms are likely to operate similarly in the context of SC therapeutics. Ultimately this knowledge will help to propose novel strategies to mitigate the allogenic barriers. Immunogenetics selection of the donor cell and immunomonitoring are key factors to allow the implementation of regenerative stem cell in the clinics.     

人羊膜间充质干细胞生物学特征及向多巴胺能神经元样细胞的分化

背景：羊膜间充质干细胞具有类似胚胎干细胞多潜能的特点,在再生医学等多种领域的临床应用具有广泛的实用性和明确的良好前景。 然而,目前对于羊膜间充质干细胞的生物学特性和分化潜能的认识仍然了解甚少。目的：建立体外分离和纯化人羊膜间充质干细胞的方法,检测羊膜间充质干细胞的体外分化特点,确定羊膜间充质干细胞在体外诱导条件下向多巴胺能神经元样细胞分化的潜能。方法：采用胰蛋白酶和胶原酶Ⅱ分步消化法从羊膜中分离羊膜间充质干细胞和羊膜上皮细胞;采用percoll梯度离心方法对羊膜间充质干细胞和羊膜上皮细胞进行纯化;流式细胞术检测羊膜间充质干细胞的表面标志,确定羊膜间充质干细胞细胞表面抗原的表达特征;对体外培养的羊膜间充质干细胞进行成脂肪和成骨诱导,确定其多向分化的潜能;采用神经细胞条件培养体系诱导羊膜间充质干细胞向多巴胺神经细胞分化,通过免疫荧光染色和激光共聚焦荧光显微镜观察和鉴定诱导后多巴胺神经元样细胞的生成。结果与结论：从羊膜组织成功分离、纯化和培养出羊膜间充质干细胞和羊膜上皮细胞。羊膜来源的间充质干细胞不仅具有典型的间充质干细胞标志,而且保留了一些胚胎干细胞的OCT-4,SOX-2和KLF4等特有干细胞标志,可以诱导分化成为脂肪细胞和骨细胞,显示羊膜间充质干细胞保持了较为原始的胚胎干细胞的特点,具有多向分化的潜能。诱导分化之前的原代羊膜间充质干细胞表达固有的多种神经细胞标记,体外诱导后羊膜间充质干细胞可分化成为β-微管蛋白Ⅲ、神经元特异性核蛋白、酪氨酸羟化酶、胶质纤维酸性蛋白、髓鞘碱性蛋白和巢蛋白等阳性表达的多巴胺能神经元样细胞。结果表明人羊膜来源的间充质干细胞所保留的多向分化潜能和有效分化成为多巴胺能神经元样细胞的特性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Novel stem/progenitor cell population from murine tracheal submucosal gland ducts with multipotent regenerative potential

The airway epithelium is in direct contact with the environment and therefore constantly at risk for injury. Basal cells (BCs) have been found to repair the surface epithelium (SE), but the contribution of other stem cell populations to airway epithelial repair has not been identified. We demonstrated that airway submucosal gland (SMG) duct cells, in addition to BCs, survived severe hypoxic-ischemic injury. We developed a method to isolate duct cells from the airway. In vitro and in vivo models were used to compare the self-renewal and differentiation potential of duct cells and BCs. We found that only duct cells were capable of regenerating SMG tubules and ducts, as well as the SE overlying the SMGs. SMG duct cells are therefore a multipotent stem cell for airway epithelial repair This is of importance to the field of lung regeneration as determining the repairing cell populations could lead to the identification of novel therapeutic targets and cell-based therapies for patients with airway diseases.     

羊水干细胞诱导肾移植免疫耐受及对氧化应激的影响

背景：干细胞在器官移植中诱导免疫耐受、延长移植物的存活时间、减轻排斥反应的作用已成为国内外众多学者研究的热点。 目的：以供体羊水干细胞诱导异基因大鼠肾脏移植免疫耐受,探讨免疫耐受的形成机制。 方法：分离培养Wistar大鼠羊水干细胞。选择雄性近交系Wistar大鼠和SD大鼠分别作为肾移植的供、受体,共分4组：假手术组(健康纯系雄性SD大鼠)、同系肾移植组(健康纯系雄性SD大鼠作供受体)、对照组(异系移植组加生理盐水)和实验组(异系移植组加羊水干细胞输注)。建立肾移植模型后,实验组由阴茎静脉缓慢推注3×109 L-1羊水干细胞 1 mL,对照组则缓慢推注生理盐水1 mL。术后第5天检测血清中肌酐、尿素氮、白细胞介素2、干扰素γ水平及氧化应激指标,流式细胞术检测外周血CD4和CD8淋巴细胞百分比,观察移植肾的病理变化。 结果与结论：实验组大鼠血清中尿素氮、肌酐、白细胞介素2、干扰素γ及氧化应激指标和尿蛋白定量、外周血淋巴细胞亚群CD4+,CD8+百分比及CD4+/CD8+比值显著低于对照组,而肌酐清除率则显著高于对照组,肾脏病理损害程度明显轻于对照组。结果显示羊水干细胞可以诱导大鼠肾移植免疫耐受,同时还可以抑制氧化应激水平,改善肾功能,减轻肾损害。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Trisomy 20 mosaicism in amniotic fluid cell culture

Chromosomal mosaicism in cultured amniotic fluid cells presents one of the most difficult problems in prenatal diagnosis in predicting the foetal phenotype. We present a case in which trisomy 20 mosaicism was diagnosed prenatally but not confirmed in the aborted foetus.     

Self-assembled collagen-human mesenchymal stem cell microspheres for regenerative medicine

Mesenchymal stem cells (MSCs)-based therapy is a promising approach in regenerative medicine and tissue engineering. However, the outcomes of existing treatments have not been satisfactory owing to suboptimal localization to implantation site, poor viability, low engraftment efficacy and lack of functional remodeling of the delivered cells. Therefore, adopting an effective cell delivery modality is among the biggest technological challenges for successful clinical applications of MSC-based therapy. We developed a novel microencapsulation technique producing self-assembled collagen-MSC microspheres and demonstrated that these microspheres could serve as excellent cell delivery devices as they were stable, injectable and able to provide a protective, growth- and migration-supporting matrix to MSCs. We also showed that MSCs could preserve their stem cell nature upon microencapsulation and easily be localized with retained viability upon in vivo implantation. These microspheres present novel cell delivery devices with optimal biological and functional profile that may facilitate clinical applications of MSC-based therapy.     

Enhancement of amniotic fluid cell growth for genetic amniocentesis

The last two decades have witnessed a logarithmic growth in demand for prenatal diagnosis of human disease through amniocentesis. Consequently, culture turn-around-time has become the major concern of all those who are primary care providers for patients seeking help. We report a rapid method for obtaining cytogenetic results from amniocytes as early as 5 days. Early tapping and reducing the turn-around-time during cytogenetic analysis may provide an alternative to chorionic villi sampling.     

Behaviour of cell cultures from human amniotic fluid.

The growth pattern of cell cultures originating from 11 amniotic fluid specimens have been observed. From each specimen 2 to 12 primary cultures were set up. In most cases growth started simultaneously in the primary cultures originating from one sample. The primary cultures lasted from 7 to 30 days. A variation was found both between cultures from different pregnancies as well as among cultures obtained from single amniotic fluids. The growth period from setting up the cultures until harvest of the cell lines for biochemical analysis ranged from 20 to 54 days. No connexion was noticed between the time spent in primary culture and the behaviour of the cell line before harvest. The effects of two types of serum (fetal calf serum and pooled human serum) on the behaviour of the cultures were compared. The cells grown in human serum were harvested a few days before those grown in fetal calf serum. The influence of different batches of medium was also examined; no significant effect of the growth pattern was found. The appearance of epithelial-like and fibroblast-like cells in cultures from 6 specimens was observed concurrently. At the time of harvest the cell lines originating from the same amniotic specimen contained the cell types in different proportions.     

广州地区4247例羊水细胞染色体核型分析

目的探讨羊水细胞染色体异常核型与产前诊断各指征之间的联系。方法对4247例高危孕妇行羊膜腔穿刺术抽取羊水进行羊水细胞染色体核型分析。结果4247例孕妇羊水培养成功4244例,成功率99．91％。发现异常核型189例,异常核型检出率为4．45％。超声软指标异常的染色体核型异常检出率最高,占39．29％。染色体数目异常107例,占56．61％。结论羊水细胞染色体检查是常用的产前诊断检查技术,能够有效的对异常胎儿进行确诊。     

Chromosome polymorphisms in karyotypes from amniotic fluid cell cultures

Frequencies of 12 fluorescent chromosome polymorphisms were scored from Q-banded karyotypes derived from 108 midtrimester diagnostic amniotic fluid cell cultures. The most frequent variants were the bright fluorescent short arm of chromosome 13 ( P = 0.458) and the bright fluorescent marker close to the centromere on chromosome 3 ( P = 0.426). The polymorphism pattern was found to be different between the maternal (blood culture) and fetal (amniotic fluid cell culture) karyotypes in each of the 25 paired cases studied. This technique is valuable in prenatal diagnosis to exclude possible maternal cell contamination and outgrowth in cases where the amniotic fluid cell cultures reveal a female karyotype.     

Hunt for pluripotent stem cell -- regenerative medicine search for almighty cell

Regenerative medicine and tissue engineering are searching for a novel stem cell based therapeutic strategy that will allow for efficient treatment or even potential replacement of damaged organs. The pluripotent stem cell (PSC), which gives rise to cells from all three germ lineages, seems to be the most ideal candidate for such therapies. PSC could be extracted from developing embryos. However, since this source of stem cells for potential therapeutic purposes remains controversial, stem cell researchers look for PSC that could be isolated from the adult tissues or generated from already differentiated cells. True PSC should possess both potential for multilineage differentiation in vitro and, more importantly, also be able to complement in vivo blastocyst development. This review will summarize current approaches and limitations to isolate PSC from adult tissues or, alternatively, to generate it by nuclear reprogramming from already differentiated somatic cells.